Rapid immunoassay for detection of antibodies or antigens incorporating simultaneous sample extraction and immunogenic reaction

ABSTRACT

The present invention is drawn to an immunoassay capable of the rapid detection of a variety of test substances that may be present in a test sample. One feature of the invention is that extraction or isolation of the test substance occurs simultaneously with the formation of the primary antigen-test substance complex. The primary antigen-test substance complex is then captured in a solid phase format having a plurality of interstitial spaces which facilitate rapid and efficient detection. The immunoassay of the present invention works over a wide range of environmental conditions and is simple enough to be used in the absence of laboratory facilities.

The present invention is a continuation of U.S. application Ser. No.08/343,165 filed Nov. 22, 1994, now U.S. Pat. No. 5,695,928, which is acontinuation-in-part application of U.S. application Ser. No.08/165,280, filed Dec. 10, 1993, now abandoned, the disclosures of whichare incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to rapid immunoassays for the detection ofa variety of test substances.

BACKGROUND OF THE INVENTION

Immunoassays, where one or more antibodies are used to detect a testsubstance in a test sample, are widely known. The evolution ofimmunoassay methods has led to increasing sensitivity and ease of use.Despite this evolution, there remains a desire to achieve the detectionand measurement of antigenic substances more rapidly and at less cost interms of time and resources, without sacrificing sensitivity orreliability of results.

In pursuit of this desire, several forms of immunoassay have beendeveloped. Some immunoassays have been developed which rely uponmultiple antibodies, one of which may be bound to a solid material. Thisbinding has been accomplished using nitrocellulose, polystyrene beads,plastic cloth, polycarbonate filters and the like. For example, U.S.Pat. No. 4,803,154 describes a “sandwich”-type immunoassay method inwhich a hydrophobic sheet is treated to create defined and delimitedhydrophilic regions which can be used to bind antibodies throughreactive aldehyde groups. The test sample containing the test substanceis placed in contact with the treated regions having the bound antibody,which captures the test substance through an immunological reaction.Next, another antibody, to which is conjugated a means of detection, isadded to the treated region and binds to the test substance immobilizedon the sheet in the first step. Executing the means of detection allowsone to measure the presence of the test substance in the test sample.This method is described as a “rapid” method, but requires from 60minutes to 48 hours to complete, not including pre-assay samplepreparation.

Convenient formats such as “dipsticks” have also been developed. In oneexample, a “dipstick” format having a polycarbonate detection membranefused to a polyvinyl chloride sheet is used to provide an immunoassaymethod usable under field conditions. C. L. Penny et al. Journal ofImmunological Methods 123: 185-192 (1989). In this method, the testsubstance is bound to a detection membrane by immersing the dipstickinto the test sample, which must be a fluid. In a second step, thedipstick is immersed into a solution containing an antibody, to which isconjugated a means of detection, which is then immobilized byimmunological reaction with the bound antigenic substance. In the finalstep, the means of detection is executed, which entails immersing thedipstick into a solution containing the appropriate detection reagents.The assay is described as requiring more than one hour to complete, notincluding pre-assay sample preparation.

In another example of the “sandwich”-type immunoassay, a first antibodyis bound to the surface of a multiwell plate. S. Kodama et al. Journalof Immunological Methods 127: 103-108 (1990). A previously prepared testsample is then mixed with an appropriate buffer containing a secondantibody, to which is conjugated a means of detection, and the combinedsolution is placed in a well of the plate. In the final step, the meansof detection is executed, which is accomplished by adding theappropriate detection reagent to the well. The presence of theantibody-test substance sandwich is then determined. The assay asdescribed requires 50 minutes for completion. Sample preparation time isnot included.

Other immunoassays are also known which purport to be rapid andeconomic, e.g., U.S. Pat. No. 4,962,023, U.S. Pat. No. 5,169,757, butall have the limitation of requiring at least one hour to complete theassay, without including the time to prepare the test sample.

Immunoassays that require less time are also known, such as the commonlyavailable latex agglutination tests for pregnancy based on detection ofhuman chorionic gonadotropin (hCG). One example of such a test is theB-Clone® hCG Assay manufactured by Monoclonal Antibodies and distributedby Baxter Scientific Products (McGaw, Ill.). Such tests are simple andrequire from a few minutes or less up to 30 minutes for detection ofhCG. These tests are designed to use urine as the test sample, whichdoes not require any preparation prior to detection of the testsubstance. In cases where the test sample containing the test substanceis more complex, such as whole blood, feces, and plant or animaltissues, test sample preparation can be a separate and time consumingstep, which can increase the time and labor needed to obtain reliableand accurate results. In these circumstances, an immunoassay that can becompleted in a similar period of time, and that combines the steps ofextraction with those of detection of the test substance would providean advantage in both time and labor.

Some immunoassays have also been developed that require only a singleincubation step. Such immunoassays are known as “simultaneous”immunoassays. One example of such an assay is described in U.S. Pat. No.4,376,110. In such an assay, an antibody bound to a solid phase supportis incubated with the test sample simultaneously with another antibodyhaving a means of detection conjugated to it. Another type ofimmunoassay described in U.S. Pat. No. 4,376,110 is a “reverse”immunoassay, which involves the stepwise addition to the liquid testsample of first the antibody having a means of detection conjugated toit followed by the addition of an antibody bound to a solid phasesupport. Such immunoassays provide for ease of handling but suffer dueto the potential presence of interfering substances in an unpreparedtest sample. Furthermore, a liquid test sample is required as theantibody-antigen reaction occurs in the liquid phase in such aimmunoassay. Such “simultaneous” or “reverse” immunoassays also requirean additional separation and wash steps to remove the capturedantibody-test substance complex from the liquid test sample. Furtherexamples of “simultaneous” and “reverse” immunoassays can be found inU.S. Pat. No. 5,011,771, where the need to prepare a test sample priorto immunoassay is ignored and the need to separate the capturedantibody-test substance complex from the liquid test sample requires anadditional sedimentation and centrifugation step. Hence, the necessityof additional steps which can require laboratory equipment can limit theusefulness of such methods for conducting immunoassays undernon-laboratory conditions.

Another immunoassay has been described in U.S. Pat. No. 5,169,789 forbacteria and viruses which can be conducted in a short period of timeand includes simultaneous lysing of the test sample and antibodyreaction. This immunoassay uses a nitrocellulose membrane having asubmicrometer pore size where the flow of the liquid containing the testsubstance from the sampling means to the capture membrane is limited todiffusion facilitated by an optional underlying absorbent layer. Such animmunoassay cannot be physically arranged to allow simultaneous visualdetermination of detection results for multiple test substances in asingle assay because of the limited flow rates and opaque nature of themembrance material. Other immunoassays based on nitrocellulose withsubmicrometer-sized pores, such as those diclosed in U.S. Pat. No.4,366,241 and U.S. Pat. No. 5,006,464, have similar shortcomings.

The present invention provides an immunoassay which is sensitive,reliable and economic and requires as little as 60 seconds to complete,including time to prepare the test sample and execute the means ofdetection of the test substance or substances.

BRIEF DESCRIPTION OF THE FIGURE

FIG. 1 shows a diagram of one embodiment of the present invention whereindividually prepared solid phase formats are placed in a multi-layeredarrangement for the capture and detection of a primary antibody-testsubstance complex while simultaneously performing both a positive andnegative control test. The solid phase formats are embedded inside aconventional plastic pipet tip such that they are appressed tightlyagainst the entire inner circumference of the tip.

(A) Solid phase format for capture of primary antibody-test substancecoinplex; (B) Solid phase format as positive control; (C) Solid phaseformat as negative control.

SUMMARY OF THE INVENTION

The present invention is drawn to an immunoassay capable of the rapiddetection of a variety of test substances that may be present in a testsample. One feature of the invention is that extraction or isolation ofthe test substance occurs simultaneously with the formation of theprimary antigen-test substance complex. The primary antigen-testsubstance complex is then captured in a solid phase format having asignificant measurement in three dimensions to form a substantial volumewith a plurality of interstitial spaces which facilitate rapid andefficient detection. Also provided is an immunoassay having solid phaseformats in a multilayered arrangement that allows the simultaneousdetection of multiple test substances in a single test sample. Thismulitlayered arrangement may also be used for the semi-quantitativedetermination of the quantity of a test substance present in a testsample. The immunoassay of the present invention works over a wide rangeof environmental conditions and is simple enough to be used in theabsence of laboratory facilities. The invention can be used for thedetection of proteins, pathogens, specific antigens, specificantibodies, haptens, chemicals in the environment, or any othersubstance for which an antibody can be obtained. The unique methodencompassed by this invention retains the sensitivity of detection ofmore elaborate methods without the accompanying cost in resources ortime. Kits for carrying out the immunoassay are also described.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to an immunosorbent assay that can be usedto rapidly detect a test substance in a desired test sample. The testsubstance may be a wide variety of molecules or compounds whichincludes, but is not limited to, antibodies, antigens, haptens, organicchemicals, proteins, enzymes, hormones, carbohydrates, lipids,macromolecules, polymers, cells of plants, insects, mammals or anycomponent thereof, viruses, virus subunits, toxins, pharmaceuticals,allergens, microorganisms such as bacteria, fungi, yeast, mycoplasma orany component thereof, or any test substance which is, or can be made tobe, immunologically reactive.

A component is any cellular or subcellular fraction prepared or derivedfrom any unicellular or multicellular organism. For example, a crude orpurified preparation of cellular membranes would be a subcellularfraction and is, or can be made to be, immunologically reactive.Likewise, a preparation of cells can be made from isolated tissue andwould be a cellular fraction which is, or can be made to be,immunologically reactive. A component may also be a subcellular fractionderived from isolated tissue, without the need of an intermediatecellular fraction step. Furthermore, a component may be a chemical orbiochemical fraction of a cell or tissue, either purified or crude,which is, or can be made to be, immunologically reactive.

The immunosorbency in the assay is provided by multiple antibodies orantigens which recognize the test substance, creating a “sandwich” orcomplex having said test substance as a central component. An antibodyis a member of the family of proteins called immunoglobins, which canspecifically combine with an immunogen. Immunoglobulins are a diversegroup of proteins and are found in mammals as class isotypes called IgG,IgM, IgA, IgD and IgE. Antibodies commonly have a variable region, towhich an antigen binds, and a constant region which is isotype andspecies specific. An antigen is defined as the substance or an integralpart of a substance which is immunologically recognized by the variableregion of the antibody and consequently binds to it.

The first step in the immunoassay of the present invention involvesextraction of the test substance from a test sample using the ExLISAbuffer system, with simultaneous formation of a primary antibody-testsubstance complex. The primary antibody is the first ligand to beimmunologically bound to said test substance. The primary antibody has ameans of detection conjugated to it. Various means of detection areknown in the art and will be discussed later.

The second step in the immunoassay of the present invention involves thephysical capture of the primary antibody-test substance complex by anappropriate affinity ligand bound to a solid phase format. Anappropriate affinity ligand depends upon the nature of the testsubstance and is generally capable of immunological binding to the testsubstance. Depending on the nature of the test substance, theappropriate affinity ligand may be either a secondary antibody (tocapture a complementary antigen or another, complementary antibody) oran antigen (to capture its complementary antibody). In the presentinvention the solid phase format is porous and has a significantmeasurement in three dimensions to form a substantial volume, whichpossesses a plurality of interstitial spaces. The appropriate affinityligand is bound to the solid phase format. “Solid phase format” is usedto mean the physical organization or arrangement of the matrix thatconstitutes the capture media of the immunoassay. Optionally, a tertiaryantibody may be added when the test substance to be detected in theimmunoassay is an antigen. This tertiary antibody would be recognizedimmunologically by the secondary antibody bound to the solid phaseformat. The tertiary antibody would also immunologically recognize thetest substance. Solid phase capture formats prepared in this way haveincreased sensitivity and consequently have greater levels of detectionthan solid phase capture formats prepared without the tertiary antibody.

The third step in the immunoassay of the present invention involves thedetection of the primary antibody-test substance complex which has beencaptured by the appropriate affinity ligand(s) attached to the solidphase format. This is afforded by the means of detection conjugated tothe primary antibody, which remains associated with the affinityligand-test substance-primary antibody complex which is formed uponphysical capture.

The rapid detection afforded by the present invention is achieved inpart by performing simultaneously both, 1) extraction of the testsubstance from the desired test sample, and 2) binding of said testsubstance to the primary antibody.

In order to achieve the combined utility of allowing for both extractionof the test substance from the test sample and formation of the primaryantibody-test substance complex to occur simultaneously it was necessaryto create a buffer system which balances the strength required of anextraction method with the delicacy of antibody-antigen binding. Theterm “ExLISA” is used to refer to this unique buffer system whichcombines the components necessary for extraction of a test substancefrom a test sample with the components necessary to perform animmunoassay similar to that found with an ELISA. The development of theExLISA buffer system overcomes the obstacles present with typicalextraction buffers which are inhibitory to antibody-antigen binding,such as the inclusion of sulfhydryl reducing agents like dithiothreitol(DTT). Furthermore, the ExLISA buffer system overcomes the additionalproblem present with typical immunoassay buffers in that they do noteffect a satisfactory test sample extraction. The immunoassay procedureis therefore accentuated by the ExLISA buffer system which allows theprimary antibody-test substance binding reaction to occur in much lesstime than with conventional methods. In most cases, the completeimmunoassay, including test sample extraction, can be completed in aboutone minute. Because extraction of the test substance from the testsample and formation of the primary antibody-test substance complexoccur simultaneously, the resulting ExLISA buffer system has a fasterreaction time than the typical immunoassay buffer system and is onlylimited by the avidity of the antibodies employed.

The ExLISA buffer system of the present invention is capable of bothoptimal extraction of the test substance to be detected from a testsample and optimal primary antibody-antigen binding in a single step.The ExLISA buffer system comprises a buffer to control pH, a detergent,a salt, a chelator, a stabilizing agent, a phenolic inhibitor, aprotease inhibitor, and a protein which is not recognized by any of theantibodies used in the assay.

The buffer selected for the ExLISA buffer system must be suitable forboth extraction of the test substance from the test sample and optimalformation of the primary antibody-test substance complex. Preferredbuffers are of the inorganic type which maintain pH between about 6.5and about 9.0, preferably about 7.5 to about 8.5. Optimal primaryantibody-test substance complex formation occurs within this range, andthe solubility of most test substances is also optimal within thisrange. Preferred buffers include sodium carbonate, sodium borate orTris-saline. The preferred range of concentrations of the buffer arefrom about 10 mM to about 200 mM, more preferably about 25 to about 100mM, and should be sufficient to provide adequate buffering capacitywithout being too high and inhibiting formation of the primaryantibody-test substance complex. In one preferred embodiment, the bufferis 50 mM sodium carbonate at pH 8.5.

A suitable detergent is added to the ExLISA buffer system to facilitatethe dissolution of cellular material during the extraction process. Theconcentration must be carefully chosen so that the dissolution ofcellular material is balanced against the denaturing effects of thedetergent, and is preferred to be in the range of about 0.01 to about0.2% (w/v), more preferably in the range of about 0.01% to about 0.1%(w/v). In addition, a preferred detergent is of the non-ionic type so asnot to inhibit the formation of the primary antibody-test substancecomplex. Suitable detergents maybe selected from TRITON® X-100 non-ionicdetergent (polyoxyethylene ether), TWEEN®9 20 non-ionic detergent(polyoxyethylene sorbitan), NP-40 (nonylphenoxy polyethoxy ethanol orMEGA-8 (octanoyl-N-methylglucamide). In one embodiment of the invention,the detergent is TRITON® X-100 at 0.05% (w/v).

An appropriate concentration of salt is chosen for inclusion in theExLISA buffer system such that it provides optimal formation of theprimary antibody-test substance complex. It is preferred that aphysiological saline solution be used at a concentration ranging fromabout 25 mM to about 175 mM, more preferably from about 100 mM to about175 mM. Preferred salts may be selected from sodium chloride, potassiumchloride and the like. In one embodiment of the invention, the salt issodium chloride at a concentration of 140 mM.

A chelator should also be used in the ExLISA buffer system in order toremove multivalent ions having a charge of positive-2 or greater fromsolution released by the extraction of the test substance from the testsample. Multivalent ions removed by said chelator that may be present inbiological or environmental test samples are Ca²⁺, Mg²⁺, Zn²⁺, Al³⁺,etc. Removal of the multivalent ions prevents protein precipitation inthe solution. It is preferred that the chelator be present in aconcentration ranging from about 0.5 mM to about 10 mM, more preferablyfrom about 1 mM to about 5 mM. An appropriate chelator may be selectedfrom EDTA (ethylenediaminetetraacetic acid), EGTA (ethyleneglycol-bis(β-amino-ethyl ether)N,N,N′, N′-tetraacetic acid), etc. In oneembodiment of the invention, the chelator is EDTA at 5 mM.

A stabilizing agent is added to the ExLISA buffer system to maintain thestability and activity of the primary antibody-test substance complexand the detection means which is conjugated to the primary antibody. Theconcentration of the stabilizing agent is critical so as not to slowdown the formation of the primary antibody-test substance complex.Preferred stabilizing agents are agar, agarose, polyethylene glycol,glycerol, ethylene glycol, etc. The preferred concentration ofstabilizer is from about 0.01% to about 20% (w/v), more preferably fromabout 1% to about 10% (w/v). In one embodiment of the invention thestabilizing agent is polyethylene glycol (3 kilodalton) at 1% (w/v).

A phenolic compound inhibitor is added to the ExLISA buffer system toremove phenolic compounds that are released into solution uponextraction of the test substance from the test sample. Phenoliccompounds will precipitate proteins, interfering with dissolution of thetest substance as well as with the formation of the primaryantibody-test substance complex. Preferred phenolic compound inhibitorsinclude the insoluble, high molecular weight, cross-linked form ofpolyvinylpyrrolidone known as polyvinylpolypyrrolidone (PVPP), sodiumborate or polyethylimine. A preferred concentration of phenolic compoundinhibitor is from about 0.01% to about 1% (w/v), more preferably fromabout 0.05% to about 0.5% (w/v). In one embodiment of the invention,PVPP is used at a concentration of 0.15% (w/v).

A protease inhibitor is added to the ExLISA buffer system in order toprevent digestion of proteins by proteases that are released byextraction of the test substance from the test sample. Such proteasescould destroy the primary antibody as well as proteinaceous testsubstances. Protease inhibitors which inhibit either serine-, cysteine-,or aspartate-type proteases are suitable for inclusion in the ExLISAbuffer system. Suitable protease inhibitors are PEFABLOC®4-(2Aminoethyl-benzenesulfonyl fluoride hydrochloride PMSF(phenylmethylsulfonyl fluoride), TLCK (N-tosyl-L-lysine chloromethylketone), TPCK (N-tosyl-L-phenylalanine chloromethyl ketone), α-caproicacid, leupeptin, benzamidine, antipain or pepstatin. The preferredconcentration of protease inhibitor is from about 0.01 mM to about 1 mM,more preferably from about 0.1 mM to about 0.5 mM. In one embodiment ofthe invention, PEFABLOC® protease inhibitor (Boehringer Mannhein) at aconcentration of 0.5 mM is used.

A protein which is not recognized by any of the antibodies used in theassay is also present in the ExLISA buffer system. This unrecognizedprotein allows the immunological blocking of non-specific antibodybinding by molecules or compounds released by extraction of the testsample. This said unrecognized protein also provides further protectionof proteins against protease attack by being exogenously supplied in ahigher concentration than other proteins released by extraction of thetest sample. Suitable unrecognized proteins are bovine serum albumin(BSA), ovalbumin, casein or fetal bovine serum. Preferred concentrationsof unrecognized protein range from about 0.05% to about 5% (w/V), morepreferably from about 0.1% to about 2% (w/v). In one embodiment of theinvention,BSA is added at 0.5% (w/v).

It is desirable for maximum convenience, economy of antibody use andrange of applications that the test sample be extracted with the ExLISAbuffer in a minimal volume. Thus it is recognized that the extractioncan be carried out in a small test tube, centrifuge tube, or anysimilarly small vessel. The extraction of the test substance from thetest sample can be facilitated by the use of a glass rod, pestle,vortexing, sonication, homogenization or any other physical force whichenhances destruction of the integrity of the test sample. In addition,glass beads, carborundum, silica or any other coarse material may beadded to further enhance the breakdown of test sample integrity. In oneexample, a plastic microfuge tube and matching pestle combination, whichhas been widely used for tissue maceration and extraction, has beenfound suitable for use in the present invention.

Another unique feature of the present invention is the solid phaseformat used to capture the primary antibody-test substance complexformed during extraction of the test substance from the test sampleusing the. ExLISA buffer. “Solid phase” as used in the art generallymeans a non-porous surface of a microtiter plate or tube wall as well asthe marginally porous surface of nitrocellulose. Hence, the solid phaseof an immunoassay in the art essentially describes a capture media witha two dimensional character. “Solid phase format” as used herein meansthe physical organization or arrangement of the matrix that constitutesthe capture media of the immunoassay of the present invention. This term“solid phase format” is therefore used herein to distinguish the presentinvention from those uses of “solid phase” present in the art.

Since it is preferred that the test sample be extracted with a minimalquantity of ExLISA buffer, a solid phase format is required that issuitable for the efficient capture of the primary antibody-testsubstance complex. It is further desired that the solid phase formatused must have a significant measurement in three dimensions to form asubstantial volume, thus providing a large effective surface area withina plurality of interstitial spaces through which the extract of the testsample can be drawn. In addition, the solid phase format used as thecapture media for the primary antibody-test substance complex should besufficiently porous so that liquids flow actively and quickly throughthe entire volume. This requires that the solid phase format have a poresize in the micrometer range or greater, as opposed to the submicronrange. This highly porous, three-dimensional “format” allowsexceptionally effective capture of the primary antibody-test substancecomplex in the rapid immunoassay of the present invention. A furtheradvantage of the porosity and physical arrangement of the solid phaseformat is that, particularly in the tip arrangement (see FIG. 1), thereis no need for the use of a filtration or centrifugation step to effectremoval of particulates, etc., which may be formed in the extraction oftissue that can clog the submicrometer-sized pores of conventional solidphases.

Many materials are available which can be fashioned accordingly and usedas appropriate solid phase formats for the present invention. Some ofthese materials are cellulose acetate, polyester coated withpolystyrene, cellulose, nitrocellulose and nylon. In one preferredembodiment, the ACT-10® cylindrical filter composed of cellulose acetatefibers and available from Hydros, Inc., Falmouth, Mass., is used as thesolid phase format. The ACT-10® is a cylindrical shape with a diameterof approximately 4 mm and a height of approximately 6 mm, with a poresize of approximately 20-30 micrometers. Several examples are givenbelow in which the ACT-100® is used as the solid phase format.

In another preferred embodiment, the cylindrical filter composed ofpolyester coated with polystyrene and available from American Filtrona,Richmond, Va. is used as the solid phase format. This filter is alsocylindrical in shape with a diameter of approximately 4 mm and a heightof approximately 6 mm, with a pore size of approximately 20-30micrometers. This solid phase format can be superior to celluloseacetate for certain immunoassays since the polystyrene coating providesan increase in the quantity of functional groups used for covalentbinding. The polyester coated with polystyrene also possesses a muchincreased physical integrity. In a further advantage over solid phaseformats composed of cellulose acetate, the use of polyester allows forantibody to be covalently bound, dried with a cryoprotectant, and thenstored dry until use. Cellulose acetate, which expands and contracts dueto moisture availability, must remain in liquid at all times oncecovalently bound with antibody. When using the polyester coated withpolystyrene, the prepared solid phase format can be stored under dryconditions for 15 months. The polyester coated with polystyrene solidphase format has been employed in all of the arrangements and assaysmentioned herein, producing equal or better assay results than thoseobtained with cellulose acetate.

The selected solid phase format must be prepared for use in theimmunoassay of the present invention by binding the appropriate affinityligand to it. It is the binding of the affinity ligand to the solidphase format which provides the capacity to capture the primaryantibody-test substance complex. In one embodiment of the invention, theappropriate affinity ligand is bound to the solid phase format by firstcreating amino binding groups through alkylation on the solid phaseformat. Suitable alkylating agents are aldehydes and N-maleimides. Oncethe solid phase format is prepared a secondary antibody is thencovalently attached to the solid phase format through the chemicallyreactive amino group. In the case of aldehydes, a reversible Schiff baseis first formed between the aldehyde and an amino group on the secondaryantibody. This bond is then stabilized through reductive alkylation witha low concentration of sodium cyanoborohydride. The thus prepared solidphase format may then be used for the capture of primary antibody-testsubstance complexes.

Alternatively, prior to capture of the primary antibody-test substancecomplex a tertiary antibody may be immunologically bound to thesecondary antibody. The tertiary antibody is chosen such that itimmunologically recognizes the test substance, immunologically bindingto it and thereby capturing the primary antibody-test substance complex.

In another embodiment of the invention, the solid phase format is firstprepared as described above to create reactive aldehyde groups. Onceprepared, strepavidin is covalently attached to the reactive groups andreduced with a low concentration of sodium cyanoborohydride. The thusprepared solid phase format may then be used for the capture of biotinlabeled secondary or tertiary antibody which is bound to the primaryantibody-test substance complex.

In a further embodiment of the invention, the solid phase format isfirst prepared as described above. Once prepared, an appropriate antigenis covalently bound to the solid phase format through a means suitableto the chemical nature of the chosen antigen. The solid phase format canthen be used for the capture of antibodies in the extract whichrecognize the bound antigen.

It is not a requirement of the present invention that the affinityligand(s) be bound to the solid phase format in a covalent manner.Antibodies, other proteins and many antigens can be adsorbed to solidphase formats through electrostatic or other molecular forces. Solidphase formats prepared in this way can be equally effective in capturinga test substance present in the test sample. It is possible, however,that long term stability of the bond between the affinity ligand(s) andthe solid phase format may be jeopardized because such bonds are weakerthan covalent bonds.

The thus prepared solid phase format-affinity ligand complex is used tocapture the, primary antibody-test substance complex obtained throughextraction of the test sample with the ExLISA buffer system. The presentinvention provides a unique capture method when compared to existingmethods. Capture of the primary antibody-test substance complex proceedsby drawing an appropriate volume of the extract through the interstitialspaces of the solid phase format which has bound to it the appropriateaffinity ligand(s) used in the immunoassay of the present invention.Drawing an appropriate volume of the extract through the large effectivesurface area afforded by the porous solid phase format providesexceptionally effective capture of the primary antibody-test substancecomplex.

In one embodiment of the present invention, the solid phase format isembedded inside a plastic pipet tip such that the solid phase format isappressed tightly against the entire inner circumference of the tip, asdepicted in FIG. 1. This creates, in essence, a low volume“chromatographic” column. As the extract prepared from the test sampleusing the ExLISA buffer is drawn into the pipet tip using a standardpipettor, the extract is drawn through the entire volume of the solidphase format. This arrangement presents an extremely large surface areato which is bound the appropriate affinity ligand(s), allowing for aquick and dynamic capture of the primary antibody-test substance complexwhich formed during extraction of the test substance from the testsample using the ExLISA buffer system. This permits the use ofrelatively small volumes of prepared extract of less than 20 μl whilesimultaneously providing a binding capacity of approximately 10 μg ormore of test substance. This unique combination of low volume and highbinding capacity allows for a high concentration recovery of the primaryantibody-test substance complex.

The solid phase format may also be employed in both eye dropper barrelsand syringe barrels with equal results. Additionally, both the eyedropper and syringe arrangements allow for aspiration and expulsion bytheir connected bulb or plunger without the need for additionalmechanical devices. Both arrangements also allow for the use of largersolid phase formats for capture of the primary antibody-test substancecomplex, which permits the covalent binding of increased concentrationsof antibody with the consequent capture of increased amounts of testsubstance.

Another arrangement that may be employed is a 96-well microplatetemplate which has wells with open-end bottoms in the form of tinyfunnels, such as that available from Polyfiltronics, Inc., Rockland,Mass., and marketed under the name FILTAPLATE®. This multiwell plate maybe seated into a small vacuum unit which allows a vacuum to be drawnfrom the top of the microplate through the funnels and into a reservoirunder the microplate. The above described polyester coated withpolystyrene solid phase formats, which are particularly suited to thisarrangement because of their sturdy physical integrity, are placed ineach well of the multiwell plate so that the solid phase format isappressed tightly against the circumference of the inner wall of thewell. Up to 96 test samples may be handled as a single assay. Thisarrangement allows for both qualitative and quantitative measurements ofa test substance, depending upon the choice of whether a soluble orinsoluble reaction product is produced by the means of detection used(see discussion below). The qualitative version of such a rapidimmunoassay according to the present invention employs a secondary ortertiary antibody covalently bound to the solid phase format, which isthen placed in a well of the multiwell plate. The test sample is thenextracted in ExLISA buffer containing the appropriate primaryantibody-enzyme conjugate and added to the well. Vacuum is then drawnthrough the well containing the polyester filter element. The well isthen washed by vacuum and a substrate which produces an insolublereaction product is added and allowed to react with the enzyme bound tothe captured test substance complex before removal by vacuum. Theresulting calorimetric detection allows for a qualitative, visualdetermination of the presence of the test substance.

The quantitative version of a rapid immunoassay based on thisarrangement is identical to the qualitative version described above,with two exceptions. First the vacuum device which the microplate isseated into is modified to accept the addition of a second 96-wellmicroplate directly under and in line with the first, so that each wellof the first microplate is directly above the corresponding well of thesecond microplate. The second plate can be any conventional 96-wellplate as used for ELISA-type assays. After capture of the test substancecomplex by the solid phase format in the well and subsequent washing,the second plate is then placed into the modified vacuum device. Anenzyme substrate which produces a soluble reaction product is then addedto the well and allowed to react with the test substance complex beforedrawing the solution containing the reaction product into the secondmicroplate. The second microplate is then removed and placed in anymicroplate reader for quantitative analysis of the resulting solution.By performing a typical standard curve comparison in other wellscontaining similarly prepared solid phase formats, very specific andsensitive quantitative analysis can be performed quickly and reliably.This arrangement is also amenable to the use of both fluorescent markersconjugated to streptavidin which are then coupled to primary antibodylabelled with biotin and also chemiluminescent substrates for alkalinephosphatase conjugated to primary antibody. The resulting sensitivitiesare at least 1000-fold greater than those possible using the celluloseactetate solid phase format in the pipet tip arrangement.

Once one solid phase format is prepared by binding of the appropriateaffinity ligand(s), it may also be combined with other similarlyprepared solid phase formats in a multi-layer arrangement. Thismultilayered arrangement permits the detection of several differentprimary antibody-test substance complexes in a single extract. Thisarrangement also permits the inclusion of positive or negative controllayers allowing the determination of positive and negative controlresults within a single assay step. When used to detect more than onetest substance in a single assay, the ExLISA buffer system would bemodified to include a variety of conjugated primary antibodies which arespecific for the range of test substances to be detected in the testsample.

Multiple solid phase formats can be used to provide a semi-quantitativeimmunoassay by binding increasing levels of secondary and/or tertiaryantibodies to each of several solid phase formats which, for example,would correspond to low, medium and high levels of test substance.Depending on the level of test substance present in the test sample, one(low), two (medium) or three (high) prepared solid phase formats willexhibit a positive reaction according to the means of detection used.

Detection of the captured primary antibody-test substance complex, andhence determination of the presence or absence of the test substance inthe test sample, may be accomplished through a wide variety of methodsthat are well known to the art. Useful references describing the generaland specific principles of detection in immunoassays may be found in E.Harlow and D. Lane, Antibodies, A Laboratory Manual, Cold Spring HarborLaboratory, New York, 1988, or in E. T. Maggio, Ed., Enzyme-Immunoassay,CRC Press, Florida, 1980. Both citations are herein incorporated byreference.

Detection techniques can be divided into direct and indirect methods. Inthe direct method, the primary antibody is labeled by conjugating to ita means of detection; the primary antibody conjugate is then use to binddirectly to the test substance. Examples of such detection means usefulin the direct method are radioactive iodine, a variety of enzymes usingchromogenic substrates, biotin, fluorochromes, metals such as colloidalgold, or the biosynthesis of the primary antibody in the presence ofradioactive amino acid precursors. In the indirect method, the primaryantibody is not conjugated to any means of detection. Instead, itsbinding to the test substance is detected by a secondary reagent, forexample using labeled anti-immunoglobin antibodies or labeled protein A.

The choice of the means of detection depends upon the assay conditions,ease of use, availability of reagents, stability of reagents and therequired sensitivity of the assay. Preferred in the present invention isthe use of direct methods as a means of detection. As a broadgeneralization relative to available direct methods as a means ofdetection, enzyme labels offer the advantage of an instant visual resultand great sensitivity making them useful for quantitative assays but aremore difficult to use in a quantitative assay because of the need tomeasure accurately the rate of reaction. Radio-iodine labeling ofprimary antibodies can give strikingly accurate quantitative results inimmunoassay procedures but stringent safety procedures are required forits use. Fluorochromes require a means of ultraviolet light excitationand detection by flourimetry or fluorescence microscopy, although theycan provide high resolution in applications such as immunocytochemistry.Metal labels such as gold colloids has become more widely available inmany useful forms. Gold is biologically inert and has a good chargedensity, allowing it to bind readily to the charged groups on theprimary antibody.

In one preferred embodiment of the present invention, enzymes areconjugated to the primary antibody as a means of detection. Antibodiescan be readily labeled by covalent coupling to enzymes. See e.g., S.Avrameas, Enzyme Markers: Their linkage with proteins and use inimmuno-histochemistry, Histochem. J. 4: 321-330, 1972. Since enzymereactions intrinsically amplify the detection signal, such conjugatescan be exceptionally sensitive. Detection levels may be as low as thepicomolar range.

A large number of enzymes have been used to label antibodies. The mostcommonly used are horseradish peroxidase, alkaline phosphatase andβ-galactosidase. Urease and glucose oxidase are also in limited use. Theideal enzyme for use as a means of detection should be inexpensive, verystable and easily conjugated. The ideal enzyme should also have a highcatalytic activity and react with a range of substrates that yield bothsoluble products and insoluble products. In a preferred embodiment ofthe present invention, bovine intestinal alkaline phosphatase is used asthe means of detection. It should be recognized, however, that manysuitable methods of detection exist, and it is encompassed within thescope of the invention that any of several methods of detection can beused.

Conjugation of means of detection are well known in the art. Enzymes maybe conjugated to primary antibodies by any one of several commonmethods. In the glutaraldehyde method, glutaraldehyde couples to theenzyme and to the primary antibody through reactive amino groupsavailable on each protein. See e.g., S. Avrameas, Coupling of enzymes toproteins with glutaraldehyde, Immunochemistry 6: 43-52, 1969. In theperiodate method, periodate treatment of carbohydrates opens the ringstructure and allows these moieties to bind free amino groups present onthe protein. See e.g., P. K. Nakane and A. Kawaoi, Peroxidase labeledantibody: A new method of conjugation, J. Histochem. Cytochem. 22:1084-1091, 1974. Conjugation of amino groups on the enzyme may also becovalently attached to thiol groups introduced on the antibody throughreaction with SPDP (N-succinimidyl-3-[2-pyridyldithio]propionate),reduction and formation of a pyridyl disulfide bridge. A. J. Cumber, etal. Preparation of antibody-toxin conjugates, Methods Enzymol. 112: 207(1985). The preferred method of conjugation of alkaline phosphatase inthe present invention is the glutaraldehyde method.

Alternatively, the means of detection conjugated to the primary antibodycan be one of several fluorochromes. Four fluorochromes are in commonuse, fluorescein, rhodamine, Texas red and phycoerytherin. Thefluorochromes are conjugated to the primary antibody by reaction withammonium chloride in DMSO (dimethyl sulfoxide). See e.g., J. W. Goding,Conjugation of antibodies with fluorochromes, J. Immunol. Methods 13:215-226, 1976. In one preferred embodiment of the present invention, theisocyanate derivatives of either fluorescein (FITC) or rhodamine (TRITC)are used.

The substrate for enzymatic means of detection may produce either asoluble or insoluble reaction product. An insoluble enzyme reactionproduct has the advantage of remaining within the solid phase formatafter reaction. This permits visual observation of the immunoassayresults. The use of an insoluble reaction product also has the benefitof permitting layering of differently prepared formats in the presentembodiment (see FIG. 1). The insolubility of the reaction productprevents leaching of the product from one layer to the next, allowingsimultaneous assay for multiple primary antibody-test substancecomplexes or for semi-quantitative analysis. Alternatively, if thesubstrate used produces a soluble enzyme reaction product, then theproduct may be leached from the solid phase format which captured theprimary antibody-test substance complex and its presence detected by,for example, spectrophotometry, so that the amount of the primaryantibody-test substance complex present may be quantitativelydetermined.

The immunoassay of the present invention may be used to detect a widevariety of test substances present in, or originating from, plants,mammals, insects, microorganisms, soil, air, water or the environment ingeneral. The test substance may be a wide variety of molecules orcompounds which includes, but is not limited to, antibodies, antigens,haptens, organic chemicals including herbicides, proteins, enzymes,hormones, carbohydrates, lipids, macromolecules, polymers, cells ofplants, insects, mammals or any part thereof, viruses, virus subunits,toxins, pharmaceuticals, allergens, microorganisrms such as bacteria,fungi, yeast, mycoplasma or any part thereof, or any test substancewhich is, or can be made to be, immunogenically active.

The general immunoassay procedure using the present invention beginswith obtaining a test sample to be assayed for the presence or absenceof the test substance. The test sample is placed in a suitable vessel inthe presence of the combined primary antibody and ExLISA buffer systemhaving an appropriate amount and composition. The test sample may beobtained by any means suitable to the nature of the test sample. Forexample, plant tissue may be obtained by cutting, slicing, tearing,punch out or clipping. Blood samples, on the other hand, could beobtained by a simple liquid transfer step. Mammalian tissue samples maybe obtained by any of the wide variety of biopsy procedures available.

It is a requirement of the present invention that the primary antibodybe combined with the ExLISA buffer system prior to its use. If many testsamples are prepared at one time for Ad comparison it is desirable thatthe amount of the test sample be reasonably consistent from sample tosample. This is important in that both the volume of the ExLISA buffersystem used and the amount of antibody must be maintained in a suitableratio to each other in order to obtain the maximum level of sensitivityand extraction of the test substance. Furthermore, to obtain the mostprecise comparisons from test sample to test sample, roughly equivalentamounts of sample should be obtained for each assay.

Once the test sample is combined with the ExLISA buffer system plusprimary antibody it may be necessary to provide some physical force todisrupt the integrity of the test sample and facilitate extraction ofthe test substance. Some examples of means to facilitate the extractionof the test substance from the test sample are by the use of a glassrod, pestle, vortexing, sonication, or homogenization. In addition,glass beads, carborundum, silica or any other coarse material mayoptionally be added to further enhance the breakdown of test sampleintegrity. One advantage of the present invention is that the ExLISAbuffer system permits optimal reaction of the primary antibody with thetest substance immediately upon its release from the test sample duringthe extraction process. When the test sample, ExLISA buffer system andprimary antibody are chosen within the guidelines of the presentinvention the test substance in the extract can be prepared for capturewithin about 5 to about 60 seconds, more preferably within about 5 toabout 30 seconds, most preferably within about 10 to about 15 seconds.

Capture of the primary antibody-test substance complex formed in thestep above occurs by drawing the extract through the interstitial spacesof the solid phase format that has been previously prepared according tothe methods described above. Drawing an appropriate volume of theextract through the large effective surface area afforded by the poroussolid phase format provides exceptionally effective capture of theprimary antibody-test substance complex. The extract is held within theinterstitial spaces of the solid phase format for about 5 to about 60seconds, more preferably for about 5 to about 30 seconds, mostpreferably for about 10 to about 15 seconds. The extract is expelledfrom the solid phase format after the appropriate amount of time.

The solid phase format which has captured the primary antibody-testsubstance complex is then washed once with an appropriate washingbuffer. This has the effect of removing residual chemicals andnon-captured substances from the solid phase format. The washing of thesolid phase format is achieved by either drawing the washing buffer intoit and then expelling or by forcing the washing buffer through it.

After washing the solid phase format containing the captured primaryantibody-test substance complex, any reagent which is required for themeans of detection is added. The immunoassay of the present inventionmay use any of the wide variety of means of detection described aboveand known in the art. In a preferred embodiment of the invention, anenzyme is conjugated to the primary antibody as the means of detection,then the appropriate substrate should be drawn into the solid phaseformat. The substrate then reacts with the conjugated enzyme, producinga detectable product. Detection by this means is achieved generallywithin about 5 minutes or less.

In one preferred embodiment, the present invention is useful fordetermining the presence or absence of a test substance in plant tissueaccording to the following steps:

1. A sharp-capped microfuge tube is used to obtain a small sample ofplant tissue by closing the cap over the, plant part to be sampled,causing a portion of the plant part to be excised from the body of theplant and being contained in the tube.

2. Add 250 μl of the ExLISA buffer system, which was previously combinedwith the appropriate primary antibody and which is conjugated toalkaline phosphatase, to the sampling tube. Macerate the tissue forapproximately 5 to 10 seconds.

3. Draw the extract into an ACT10® solid phase format (Hydros, Inc.,Falmouth, Mass.) which has been prepared with the appropriate affinityligand(s) as described above. Hold this mixture for approximately 5 to15 seconds to facilitate capture of the primary antibody-test substancecomplex and then expel.

4. Wash the tip with 1 ml of a washing buffer (10 mM7Tris-TRIZMA pH 8.0,0.05% w/v TWEEN® 20 non-ionic detergent, 0.02% w/v sodium azide) byforcing the buffer down through the tip.

5. Draw into the tip approximately 50 μl of the enzyme substrate,nitroblue tetrazolium plus bromochloroindole phosphate.

6. Wait 5 to 10 minutes to allow for full color development and recordresults.

Alternatively, if it is desired to detect antibodies in a test sample,then the appropriate antigen would be covalently bound to the solidphase format and using the same principles of single step extraction andantigen reaction coupled with effective capture of the antigen anddetection thereof.

For example, isotyping of immunoglobulins, such as IgG or IgM, in orderto determine the antibody population within a given serum can beaccomplished rapidly with the present invention. Antibodies prepared toeach immunoglobulin class can be bound to the appropriate solid phaseformat. Individual solid phase formats could be prepared for eachimmunoglobulin class and then each one is place within the pipet tip ina multilayered arrangement (see FIG. 1). Upon simultaneous extraction ofthe serum and formation of the primary antibody-immunoglobulin complexusing an appropriate ExLISA buffer, the test sample is drawn througheach individual layer in order to capture a different primaryantibody-immunoglobulin complex within the interstitial spaces of eachindividual layer of prepared solid phase format. A standard detectionmethod such as any of those listed above is then used to indicate thepresence of different immunoglobulins in the test sample.

Another example of the use of the present invention to detect antibodiesin serum is the, detection of antibodies to human immunodeficiency virus(HIV). Either formalin-fixed HIV or coat protein is bound to anappropriate format. Serum is extracted and formation of the primaryantibody-test substance complex is accomplished using an appropriateExLISA buffer system containing, e.g., an anti-human antibody as theprimary antibody. The extract is drawn through the porous solid phaseformat to which is bound the non-viable virus or coat protein, capturingthe primary antibody-HIV-specific antibody complex. Detection thenoccurs by any of several previously described means.

The present invention, combining the elements of simultaneous extractionand primary antibody-test substance complex formation, capture of saidcomplex, and detection of said complex provides an immunoassay which canbe performed in no more than a few minutes and under a wide range ofenvironmental conditions. It is simple enough to be used in the absenceof laboratory conditions—in the field, office, greenhouse, farm, orhome. The invention can be used for the detection of expressed proteins,pathogens, specific antigens, specific antibodies, chemicals in theenvironment, or any other substance for which an antibody, eithermonoclonal or polyclonal can be obtained. The unique method encompassedby this invention retains the sensitivity of detection of more elaboratemethods without the accompanying cost in resources or time. Theimmunoassay of the present invention can be performed by any personaccording to simple instructions, allowing for use at home, office,field or any other location.

In order to maximize the utility of the present invention, it isdesirable to have the immunoassay assembled in a kit form. Such a kit isconvenient to use as it contains all the reagents necessary to obtainresults. In one embodiment of the present invention, a kit forimmunoassay is comprised of a microfuge tube for each test sample, apipet tip for each test sample having embedded in it a solid phaseformat prepared with an appropriate affinity ligand(s) for the captureof the primary antibody-test substance complex in a multilayeredarrangement along with a solid phase format prepared as a positivecontrol and a solid phase format prepared as a negative control, apestle suitable for grinding a test sample in the microfuge tube, adispensing bottle of ExLISA buffer system which optionally may or maynot have the primary antibody conjugated to a means of detectionpre-diluted in it, a dispensing bottle of washing buffer, a 1 mldisposable transfer pipet for each test sample to be used for thewashing buffer, and a dispensing bottle of an appropriate reagent to beused with the means of detection conjugated to the primary antibody.Kits containing sufficient parts for up to 20 test samples areconvenient for use under field conditions.

In another embodiment of the invention, a kit for large scale use may beprepared such that the components are prepared in bulk and placed inquantities of 20 or more. Larger volumes of reagents are supplied, orare supplied in 2-fold to 10-fold concentrations, and additionaldisposable pipets and pestles are added to the kits.

The following examples further describe the materials and methods usedin carrying out the invention and the subsequent results. They areoffered by way of illustration, and their recitation should not beconsidered as a limitation of the claimed invention.

EXAMPLES A. ExLISA Buffer

One preferred example of the ExLISA buffer system is composed of 50 mMNaCO₃ at pH 8.5, 140 mM NaCl, 5 mM EDTA, 0.05% TRITON® X-100 non-ionicdetergent, 0.15% PVPP (w/v), 1.% PEG (3 kilodalton), 0.5 mMPEFABLOC®protease inhibitor, 0.5% BSA. This example has been found to be widelyuseful for the extraction of plant tissue or cells, effectivelycombining extraction and formation of the primary antibody-testsubstance complex into one simultaneous process. The reagents arecommonly available through the indicated chemical supply houses.

  50 mM NaCO3 pH 8.5 (Sigma Chem.)  140 mM NaCl (Sigma Chem.)   5 mMEDTA (Sigma Chem.) 0.05% TRITON ® X-100 (Sigma Chem.) 0.15% PVPP (SigmaChem.)  1.0% PEG (3 kd) (Sigma Chem.)  0.5% BSA (Sigma Chem.)  0.5 mMPEFABLOC ® (Boehringer Mannheim)

Prior to use, the ExLISA buffer in this example is combined with aprimary antibody, which has conjugated to it alkaline phosphatase as oneexample of a detection means. The primary antibody is chosen such thatit immunologically recognizes the test substance and may be preparedfrom any source, for example, goat, rabbit, rat or mouse. Immunoaffinitypurification may be used to obtain purified primary antibody, and calfintestine alkaline phosphatase may be conjugated to it using a modifiedglutaraldehyde method of Avrameas, supra. The conjugated primaryantibody is titered to determine the working dilution for use in theassay according to the present invention. The appropriate dilution mustbe determined for each batch of primary antibody made, but is generallyin the range of from 1:100 to 1:1000, made in ExLISA buffer. Thisprimary antibody conjugate in ExLISA buffer instantaneously binds to thetest substance when the plant tissue or cells are macerated in itspresence.

B. Detection of Bacillus thuriaiensis kurstaki (Btk) Endotoxin Expressedin Leaf Tissue

The solid phase format, one example of which may be purchased fromHydros, Inc. (Falmouth, Mass.) and is known commercially as ACT-10®,which is capable of binding primary amine groups present on proteins, asdescribed above. The secondary antibody covalently bound to the solidphase format was immunopurified goat anti-rabbit antibody (commerciallyavailable from a multitude of vendors) at a 100 μg/ml concentration in aborate-saline (100 mM boric acid, 25 mM sodium borate, 75 mM sodiumchloride, pH 8.5) buffer at 4 C overnight. Unbound goat anti-rabbitantibody was then removed from the solid phase format and bound goatanti-rabbit antibody was covalently attached to the format by reductionof the Schiff's base with 20 mM sodium cyanoborohydride for 2 to 4hours. Any remaining active sites on the solid phase format were thenblocked with blocking buffer (3% w/v bovine serum albumin, 0.02% w/vsodium azide, 10 mM sodium phosphate, 140 mM sodium chloride, pH 7.5)for at least 1 hour at 4 C. The blocking buffer was then removed andimmunoaffinity purified rabbit anti-Btk tertiary antibody was added at,15 μg/ml in diluent buffer (1% w/v bovine serum albumin, 0.02% sodiumazide, 0.05% TWEEN® 20 non-ionic detergent, 10 mM sodium phosphate, 140mM sodium chloride, pH 7.5) and incubated at 4 C overnight. The tertiaryantibody binds to the secondary antibody in a normal antibody-antigenreaction.

Three layers of solid phase format were embedded in a pipet tip (asshown in FIG. 1) for the Btk endotoxin protein assay. The bottom solidphase format layer was an assay positive control which consisted of calfintestine alkaline phosphatase covalently attached to the solid phaseformat. The solid phase format layer above the positive control layer isthe negative control which consists of adsorbed bovine serum albumin.The top solid phase format layer is the layer used to capture theprimary antibody-test substance complex from the extract, which isdescribed above. The three layers of solid phase format embedded in thepipet tip were stored in tip storage buffer (25 mM Tris, 140 mM NaCl, pH7.8, 2% w/v BSA, 0.05% w/v TWEEN® 20 non-ionic detergent, 0.02% w/vNaN₃) until the test sample extract was prepared (can be stored up to 6months). The storage buffer was removed by aspiration from the preparedsolid phase format immediately prior to its use.

The leaf tissue of the test plant was obtained by snapping the lid of a1.5 ml microfuge tube on a leaf, which removes a small circular area oftissue. This test sample was kept cool when the assay could not beperformed within one hour, or if the ambient temperature was above 90°F. This tissue was then macerated in the presence of 250 μl of theExLISA buffer described in Example A, above, using a Kontes® (Vineland,N.J.) plastic pestle for maceration by a firm rotating action forapproximately 5 seconds. During extraction, the primary goat anti-Btkantibody binds to the Btk endotoxin protein released from the tissue bymaceration with the pestle in the microfuge tube. The extract was thendrawn through the interstitial spaces of the prepared solid phaseformat, allowing the primary antibody-Btk complex to be captured by thesecondary antibody-tertiary antibody complex bound to the solid phaseformat. The extract was held in the solid phase format for at least 15seconds and then expelled to a waste vessel. The extract may remain inthe solid phase format for up to one hour at room temperature beforeexpelling, although beyond 2-3 minutes the sensitivity only increasesmarginally. After capture the solid phase format was washed with 1 ml ofwash buffer (10 mM Tris-TRIZMA pH 8.0, 0.05% w/v TWEEN® 20 non-ionicdetergent, 0.02% w/v sodium azide). The solid phase format was thensaturated with nitroblue tetrazolium plus bromochloroindole phosphate,which is a single component substrate for alkaline phosphatase availablefrom the chemical supply house Kirkegaard & Perry. Depending upon theamount of Btk endotoxin protein present, the reaction may begin almostimmediately. However, generally the reaction was allowed to proceed forat least 5 minutes before results were recorded. Only the full sandwichcomplex will be identified by the substrate, which is specific foralkaline phosphatase. Care must be taken to keep the tip with the enzymesubstrate in it away from UV light as the substrate is light sensitive.

The result for a particular test sample was determined by recording thecolor present in each of the three layers. The bottom solid phase formatlayer, as the positive control, should always turn dark blue color whenthe assay is performed. The solid phase format layer above the positivecontrol is the negative control and should always remain white toslightly gray in color when assayed. The top solid phase format layer isfor capturing, and subsequently detecting, the presence of the primaryantibody-Btk protein complex and will turn color only in its presence.Therefore, a dark-blue unknown solid phase format represented a positiveplant for BT presence and no color change (white, or slightly gray asdictated by the negative control) represented a BT negative plant. Ifthe positive control (bottom solid phase format layer) does not turndark blue or the negative control (middle solid phase format layer) doesnot remain white or light gray, then the assay MUST be repeated as thisindicates a false reading.

Over 500 plants have been tested for the presence of the Btk endotoxinby both the rapid immunoassay of the present invention under fieldconditions and by conventional laboratory-based ELISA. A perfectcorrelation was observed between the two assays, showing that the rapidimmunoassay is as sensitive and reliable as the more time-consuming andresource-intensive ELISA.

C. Detection of Cucumber Chitinase in Leaf Tissue

This immunoassay follows the same procedure as described above with theexception of the antibodies used. To assay for the presence of theexpressed cucumber chitinase protein in leaf tissue, goat anti-mousesecondary antibody was covalently bound to the desired solid phaseformat by Shiff's base formation with aldehyde groups, reduction bycyanoborohydride and blocking of unreacted sites with bovine serumalbumin. A mixture of three immunopurified monoclonal mouse tertiaryantibodies against cucumber chitinase were bound to the goat anti-mousesecondary antibody by way of a normal antibody-antigen reaction. Thisprepared solid phase format was then used to capture the primaryantibody-test substance complex. The prepared solid phase format wasembedded into a pipet tip, along with a solid phase format layer for thepositive control and a separate solid phase format layer for thenegative control, as described above. The ExLISA buffer system was thesame as described in Example A, above, except that immunoaffinitypurified rabbit anti-cucumber chitinase primary antibody was added toit. This primary antibody was conjugated to alkaline phosphatase asdescribed above. The extract prepared by macerating the tissue in thepresence of the ExLISA buffer system was then drawn into theinterstitial spaces of the prepared solid phase format. Upon treatmentwith the enzyme substrate, the upper solid phase format layer turneddark blue when the primary antibody-chitinase protein complex waspresent.

Using the principles described in Example D, below, the expression ofchitinase has also been detected in a semi-quantitative manner using amulti-layered arrangements of solid phase formats prepared withincreasing levels of secondary antibody. Pr-1 basic protein, with isoften endogenously expressed in plants concomitantly with chitinase inresponse to pathogen infection, was determined simultaneously withchitinase as described above when solid phase formats prepared with theappropriate secondary antibody were combined in a tip arrangement.

D. Detection of Fungal Pathogens of Wheat

This immunoassay follows the same procedure as previously described withthe exception the antibodies used for detection of the pathogen.Immunoaffinity purified goat anti-sheep secondary antibody was bound tothe appropriate solid phase format through reactive aldehyde groups, theSchiff's base was reduced by sodium cyanoborohydride, and unreactedsites were blocked by the addition of bovine serum albumin. Purifiedsheep tertiary antibody against Septoria tritici was bound to the goatanti-sheep secondary antibody by way of a normal antibody-antigenreaction. The ExLISA buffer system was the same as described in ExampleA, above, except that immunoaffinity purified sheep anti-Septoriatritici conjugated to alkaline phosphatase was added to it. The extractprepared by macerating the tissue in the presence of the ExLISA buffersystem was then drawn into the interstitial spaces of the prepared solidphase format. Upon treatment with the enzyme substrate, the upper solidphase format layer turned dark blue when the primary antibody-Septoriatritici antigen complex was present.

Semi-quantitative detection of the level of Septoria tritici or Septorianodorum infestation of wheat plants was also achieved using the presentimmunoassay by adjusting the levels of the secondary and/or tertiaryantibodies covalently bound to the solid phase format. Each of threesolid phase formats were prepared with decreasing levels of secondaryantibody specific for either Septoria tritici or Septoria nodorum,depending on the species of pathogen detected. The quantity of secondaryantibody which corresponds to low, medium, or high infestation levelswas pre-determined by a sandwich ELISA using the same antibodies inorder to calibrate the rapid immunoassay. The three prepared solid phaseformats, along with solid phase formats prepared as positive andnegative controls, were placed in a conventional plastic pipet tip in astacked and contiguous manner similar to that shown in FIG. 1. A leafextract was prepared in the ExLISA buffer system containing a primaryantibody-enzyme conjugate where the primary antibody was specific foreither Septoria tritici or Septoria nodorum, again depending on thespecies of pathogen detected. The extract containing the test substancewas drawn through the five stacked and contiguous solid phase formats asprepared above. The extract was expelled, the tip was washed, and enzymesubstrate was drawn into the tip to allow for visual assessment.Differing levels of Septoria resulted in either one, two or threeprepared solid phase formats becoming colorimetrically positive. Inother words, a low level of Septoria infestation resulted in oneprepared solid phase format being positive, a medium level ofinfestation resulted in two formats being positive and a high levelresulted in three formats being positive.

Both Septoria tritici and Septoria nodorum were also be detectedsimultaneously in the same assay from the same test sample. By using twoprepared solid phase formats, one covalently bound with a secondaryantibody to detect one pathogen and the other covalently bound withsecondary antibody to detect the other pathogen, it was possible todistinguish if one or both pathogens were present. Both prepared solidphase formats were placed in a single pipet tip, along with solid phaseformats prepared as positive and negative controls, similar to thatdepicted in FIG. 1. The leaf sample was homogenized in the presence ofExLISA buffer and two primary antibody-enzyme conjugates, eachrecognizing one of the two Septoria species. The test sample extract wasdrawn through the tip, expelled, the tip was washed, and then enzymesubstrate was drawn into the tip for calorimetric visualization. Whenboth species of Septoria were present in the test sample, both the solidphase formats prepared with secondary antibody reacted positively. Whenonly one species of Septoria was present in the test sample, then onlythe solid phase format prepared with the secondary antibody recognizingthat species reacted positively.

Detection and semi-quantitation of Pseudocercosporella herpotrichoides(Eyespot disease in wheat) can also be accomplished using the rapidimmunoassay. The immunoassay uses polyclonal antibodies specific for P.herpotrichoides as the primary antibody with an enzyme conjugated to itas a means of detection and as the secondary antibody covalently boundto solid phase formats. Wheat tissue is homogenized in ExLISA buffercontaining the antibody-enzyme conjugate and is drawn through theprepared solid phase formats as outlined above. Levels of Eyespotdisease infestation can be semi-quantitated within a single assay asdescribed above for Septoria, or the presence of P. herpotrichoides, S.tritici, and S. nodorum can all be detected within a single rapidimmunoassay.

E. Detection of Fungal Pathogen (Sigatoka) of Banana

This immunoassay will be exactly the same as described in Example D,except with appropriate antibodies. Both monoclonals and polyclonals areused for the primary, secondary and tertiary antibodies, with solidphase format preparation, capture of the primary antibody-test substancecomplex and detection with alkaline phosphatase as the means ofdetection.

F. Detection of Insect Gut Receptors

The purpose of this immunoassay is to determine the absence or presenceof specific toxin binding receptors found in the gut membrane of asingle individual of the insect Plutella. Each toxin has a specificbinding receptor in the gut membrane. This immunoassay follows a similarprocedure as that described above with the exception that a purifiedspecific endotoxin isolated from Bacillus thuringiensis is bound to aappropriate solid phase format through interaction with an aldehydegroup, the Schiff's base is reduced by sodium cyanoborohydride, and theunreacted sites are blocked by the addition of bovine serum albumin. TheExLISA buffer system is prepared with immunoaffinity purified rabbitprimary antibody which immunologically recognizes Plutella brush bordermembrane vesicles. The primary antibody is conjugated to alkalinephosphatase. A single insect is placed in a microfuge tube and maceratedwith a pestle in the ExLISA buffer system containing the primaryantibody. The resulting extract is drawn into the interstitial spaces ofthe prepared solid phase format by aspiration and is captured by theBacillus thuringiensis endotoxin which is covalently bound to the solidphase format. Different Bacillus thuringiensis endotoxins are covalehtlybound to separate solid phase formats, and each separate solid phaseformat is embedded in layers in the pipet tip. Although the rabbitanti-brush border membrane primary antibody will non-specifically bindall insect gut receptors present in the extract, only those which bindthe specific endotoxin present in a specific format layer will becaptured by that layer. Therefore, only a primary antibody-gut receptorcomplex specific for the endotoxin bound in a particular format layerwill be captured by that layer, and produce a positive result withdetection by alkaline phosphatase as exhibited by the development of adark blue color in that layer. The variation in gut receptor binding fordifferent Bacillus thuringiensis endotoxins may be quickly determined byobserving which solid phase format layers give a positive reaction andwhich give a negative reaction.

G. Detection of Antibodies to Brucella abortus in Bovine Serum

Brucella abortus is a major bacterial pathogen of cattle and causesbovine brucellosis, a veterinary disease with significant economicconsequences. The disease is highly contagious and subject to governmentquarantine. A rapid means of determining the presence of the diseasewould be by detecting the presence of antibodies against the Brucellapathogen in bovine serum and would be advantageous in monitoring andcontrolling the spread of the disease. This has the benefit of detectingantibody over pathogen where the titer of the pathogen is not stable.Partially purified lipopolysaccharide from Brucella abortus or a crudeextract of Brucella abortus is prepared and adsorbed onto an appropriatesolid phase format. Lipopolysaccharide binding to the solid phase formatmay possibly be enhanced by the use of lectins. Any remaining bindingsites on the solid phase format must be blocked with a protein otherthan bovine serum albumin, such as ovalbumin or casein. The thusprepared solid phase format is embedded in a multilayered arrangementinto a pipet tip along with a separate solid phase format for a negativecontrol (middle layer, to which ovalbumin has been bound) and for apositive control (bottom layer, to which alkaline phosphatase has beenbound). An appropriate amount of bovine serum is diluted into ExLISAbuffer to which has been added goat anti-bovine immunoglobulin primaryantibody conjugated to alkaline phosphatase. The extract of the testsample thus prepared is then drawn into the interstitial spaces of themultilayered solid phase formats. After the extract is held for about 15seconds it is expelled. The pipet tip is then washed with washingbuffer. The solid phase format is then saturated with nitrobluetetrazolium plus bromochloroindole phosphate, which is a substrate foralkaline phosphatase. Depending upon the amount of antibody to Brucellaabortus present in the serum sample, the reaction may begin within oneminute. However, generally the reaction proceeds for at least 10 minutesbefore results are recorded.

H. Detection of Herbicides in Soil and Water Samples

This immunoassay follows the same procedures as described above with theexception of the antibodies used. To assay for the presence of theherbicides such as metalochlor or sulfonylurea in soil or water, themonoclonal antibodies described in U.S. Ser. No. 08/359,689, acontinuation of U.S. Ser. No. 08/035,872, filed Mar. 23, 1993, whichimmunologically recognize metalochlor, and in U.S. Ser. No. 07/988,509,filed Dec. 10, 1992, which immunologically recognize member of the classof herbicides known as sulfonylureas, are used as primary and secondaryantibodies. Secondary antibody is covalently bound to the desired solidphase format by Shiff's base formation with aldehyde groups, reductionby cyanoborohydride and blocking of unreacted sites with bovine serumalburmin. This prepared solid phase format is then used to capture theprimary antibody-herbicide complex. The prepared solid phase format isembedded into a pipet tip, along with a solid phase format layer for thepositive control and a separate solid phase format layer for thenegative control, as described above. The ExLISA buffer system was thesame as described in Example A, above, except that immunoaffinitypurified primary antibody which immunologically recognizes the herbicideto be detected is added to it. This primary antibody was conjugated toalkaline phosphatase as described above. In the case of soil samples,the herbicide must first be extracted using procedures known in the art,whereupon the extract so made is combined with ExLISA buffer. Watersamples may be used without preparation, or may be concentrated by knownmethods prior to combining with ExLISA buffer. The resulting combinedmixture is then drawn into the interstitial spaces of the prepared solidphase format. Upon treatment with the enzyme substrate, the upper solidphase format layer will turn dark blue when the primaryantibody-herbicide complex is present.

Using the principles described in Example D, above, the presence ofherbicides may also be detected in a semi-quantitative manner using amulti-layered arrangement of solid phase formats prepared withincreasing levels of secondary antibody. In addition, the presence ofmore than one herbicide in the test sample may be determinedsimultaneously as described above when solid phase formats prepared withthe appropriate secondary antibody are combined in a tip arrangement.

The extraction process using the ExLISA buffer system has proven to beas successful as various other extraction methods currently in use. Thiswas shown by direct comparisons of standard methods and the methodencompassed by the present invention. Immunoassays for Bacillusthuringiensis kurstaki endotoxin, Bacillus thuringiensis tenebrionisendotoxin, PR1 a protein, cucumber chitinase and phosphinothricinacetyltransferase produced similar quantitative values when extractswere prepared using the ExLISA buffer system as compared to thoseobtained from the use of standard extraction techniques. Hence, theextraction method using the ExLISA buffer system produces identicalresults when compared with standard methods.

All publications and patent applications mentioned in this specificationare indicative of the level of skill of those skilled in the art towhich this invention pertains. All publications and patent applicationsare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended claims.

What is claimed is:
 1. A kit for detection by immunoassay of at leastone test substance in a test sample comprising in packaged combination:(a) a primary antibody which immunologically recognizes said testsubstance wherein said primary antibody is conjugated to a means ofdetection; (b) a cylindrical solid phase format contained in acylindrical vessel, the vessel having an inner wall and at least oneopen end, said solid phase format being circumferentially and tightlypressed against the inner wall of said vessel, having a pore size ofabout 20-30 micrometers with a plurality of interstitial spaces andcapable of capturing a complex formed by said primary antibody and saidtest substance; (c) a second vessel containing an ExLISA buffer systemwhich is capable of supporting simultaneous sample extraction of saidtest substance from said test sample and antigen-antibody reactionconsisting essentially of: (i) a buffer; (ii) a non-ionic detergentpresent at a concentration of about 0.01 to about 0.2% (w/v); (iii) asalt in an amount effective to provide a physiological saline solutionand present at a concentration of about 25 mM to about 175 mM; (iv) achelator in an amount effective to remove multivalent ions; (v)stabilizing agent in an amount effective to maintain stability andactivity of an antibody in said antigen-antibody reaction and present ata concentration from about 0.01 to about 20% (w/v); (vi) a phenoliccompound inhibitor; (vii) a protease inhibitor; (viii) a protein in anamount effective to immunologically block non-specific antibody bindingand present at a concentration from about 0.05 to 5% (w/v); (d) reagentsreactive with said means of detection to produce a detectable reactionproduct; and (e) means for dispensing said reagents.
 2. The kitaccording to claim 1 wherein said buffer is selected from sodiumcarbonate, sodium borate or Tris-saline.
 3. The kit according to claim 1wherein said detergent is selected from polyoxyethylene ether,polyoxyethylene sorbitan, nonylphenoxy polyethoxy ethanol oroctanoyl-N-methylglucamide.
 4. The kit according to claim 1 wherein saidsalt is selected from sodium chloride or potassium chloride.
 5. The kitaccording to claim 1 wherein said chelator is selected fromethylenediaminetetraacetic acid or ethylene glycol-bis(β-amino-ethylether) N,N,N′,N′-tetraacetic acid.
 6. The kit according to claim 1wherein said stabilizing agent is selected from agar, agarose,polyethylene glycol, glycerol or ethylene glycol.
 7. The kit accordingto claim 1 wherein said phenolic inhibitor is selected frompolyvinylpolypyrrolidone, sodium borate or polyethylimine.
 8. The kitaccording to claim 1 wherein said protease inhibitor is selected from4-(2-Aminoethyl)-benzenesufonyl fluoride hydrochloride,phenylmethylsulfonyl fluoride N-tosyl-L-lysine chloromethyl ketone,N-tosyl-L-phenylamine chloromethyl ketone, α-caproic acid, leupeptinbenzamidine, antipain or pepstatin.
 9. The kit according to claim 1wherein said protein is selected from bovine serum albumin, ovalbumin,casein, or fetal bovine serum.
 10. The kit of claim 1 wherein said meansof detection is an enzyme.
 11. The kit of claim 10 wherein said enzymeis selected from alkaline phosphatase, peroxidase, or β-galactosidase.12. The kit of claim 10 further comprising a substrate which reacts withsaid enzyme to produce a detectable insoluble reaction product.
 13. Thekit of claim 10 further comprising a substrate which reacts with saidenzyme to produce a detectable soluble reaction product.
 14. The kit ofclaim 1 wherein said means of detection is a fluorochrome.
 15. The kitof claim 14 wherein said fluorochrome is selected from fluoresceinisothiocyanate, tetramethylrhodamine isothiocyanate, Texas red, orphycoerythrin.
 16. The kit of claim 1 wherein said test substance boundby said primary antibody is selected from a bacterium, an insect, afungus, a virus, a plant cell, a protein, a carbohydrate, a hormone, apharmaceutical, a lipid, a herbicide, or an immunoreactive componentthereof.
 17. The kit of claim 16 wherein said bacterium is Brucellaabortus.
 18. The kit of claim 16 wherein said insect is Plutella. 19.The kit of claim 16 wherein said fungus is selected from the groupconsisting of Septoria tritici, Septoria nodorum, Pseudocercosporellaherpotichoides and a fungal pathogen causing Sigatoka of banana.
 20. Thekit of claim 16 wherein said virus is human immunodeficiency virus. 21.The kit of claim 16 wherein said protein is selected fromimmunoglobulins, cucumber chitinase, or Bacillus thuringiensisendotoxin.
 22. The kit of claim 1 wherein said solid phase format isselected from cellulose acetate, polyester coated with polystyrene,cellulose, or nylon.
 23. The kit of claim 22 further comprising aplurality of solid phase formats and wherein said solid phase formatsare arranged in stacked and contiguous layers wherein each layercaptures a different primary antibody-test substance complex.
 24. Thekit of claim 22 further comprising a plurality of solid phase formatsand wherein said solid phase formats are arranged in stacked andcontiguous layers wherein each layer captures a different quantity ofsaid primary antibody-test substance complex.
 25. An immunoassay fordetection of at least one test substance in a test sample comprising thesteps of: a) extracing the test substance from said test sample with anExLISA buffer system in the presence of a primary antibody whichimmunologically recognizes said test substance to form a primaryantibody-test substance complex, wherein said primary antibody isconjugated to a means of detection; wherein said ExLISA buffer systemconsists essentially of: i) a buffer; ii) a non-ionic detergent presentat a concentration of about 0.01 to about 0.2% (w/v); iii) a salt in anamount effective to provide a physiological saline solution and presentat a concentration of about 25 mM to about 175 mM; iv) a chelator in anamount effective to remove multivalent ions; v) stabilizing agent in anamount effective to maintain stability and activity of an antibody insaid antigen-antibody reaction and present at a concentration from about0.01 to about 20% (w/v); vi) a phenolic compound inhibitor; vii) aprotease inhibitor; viii) a protein in an amount effective toimmunologically block non-specific antibody binding and present at aconcentration of about 0.05 to 5% (w/v); (b) providing a cylindricalsolid phase format contained in a cylindrical vessel, the vessel havingan inner wall and at least one open end, said solid phase format beingcircumferentially and tightly appressed against the inner wall of saidvessel, having a pore size of about 20-30 micrometers with a pluralityof interstitial spaces and further having immobilized therein asecondary antibody capable of immunologically recognizing (i) said testsubstance or (ii) a tertiary antibody which immunologically recode saidtest substance; (c) contacting the extract step (a) with (i) the solidphase format of step (b)(i) or (ii) the solid phase format of step(b)(ii) and said tertiary antibody to capture said primary antibody-testsubstance complex; (d) washing the solid phase format of step (c) with abuffer; and (e) detecting said test substance in the test sample bydetecting the presence of said captured primary antibody-test substancecomplex.
 26. An immunoassay for detection of at least one test substanceof claim 25, further comprising taking said test sample from a plant.27. The immunoassay of claim 25 wherein said test substance is selectedfrom a bacterium, an insect, a fungus, a virus, a plant cell, a protein,a carbohydrate, a hormone, a pharmaceutical, a lipid, a herbicide, or animmunoreactive component thereof.
 28. The immunoassay of claim 27wherein said bacterium is Brucella abortus.
 29. The immunoassay of claim27 wherein said insect is Plutella.
 30. The immunoassay of claim 27wherein said fungus is selected from the group consisting of Septoriatritici, Septoria nodorum, Pseudocercosvorella herpotrichoides and afungal pathogen causing Sigatoka of banana.
 31. The immunoassay of claim27 wherein said virus is human immunodeficiency virus.
 32. Theimmunoassay of claim 27 wherein said protein is selected fromimmunoglobins, cucumber chitinase, or Bacillus thuringiensis endotoxin.33. The immunoassay of claim 25 wherein said solid phase format isselected from cellulose acetate, polyester coated with polystyrene,cellulose, or nylon.
 34. The immunoassay of claim 33 further comprisinga plurality of said solid phase formats wherein said solid phase formatsare arranged in stacked and contiguous layers wherein each layercaptures a different primary antibody-test substance complex.
 35. Theimmunoassay of claim 33 further comprising a plurality of said solidphase formats wherein said solid phase formats are arranged in stackedand contiguous layers wherein each layer captures a different quantityof said primary antibody-test substance complex.
 36. The immunoassay ofclaim 25 wherein said means of detection is an enzyme.
 37. Theimmunoassay of claim 36 wherein said enzyme is selected from alkalinephosphatase, peroxidase, or β-galactosidase.
 38. The immunoassay ofclaim 36 wherein said enzyme is reacted with a substrate therefore toproduce a detectable soluble reaction product.
 39. The immunoassay ofclaim 36 wherein said fluorochrome is selected from fluoresceinisothiocyanate, tetramethylrhodamine isothiocyanate, Texas red, orphycoerythrin.
 40. The immunoassay of claim 25 wherein said means ofdetection is a fluorochrome.
 41. The immunoassay of claim 40 whereinsaid fluorochrome is selected from fluorescein isothiocyanate,tetramethylrhodamine isothiocyanate, Texas red, or phycoerythrin.
 42. Animmunoassay for detection of at least one test substance in a testsample comprising the steps of: (a) extracting the test substance fromsaid test sample with an ExLISA buffer system in the presence of aprimary antibody which immunologically recognizes said test substance toform a primary antibody-test substance complex, wherein said primaryantibody is conjugated to a means of detection, wherein said ExLISAbuffer system consists essentially of: i) a buffer; ii) a non-ionicdetergent present at a concentration of about 0.01 to about 0.2% (w/v);iii) a salt in an amount effective to provide a physiological salinesolution and present at a concentration of about 25 mM to about 175 mM;iv) a chelator in an amount effective to remove multivalent ions; v)stabilizing agent in an amount effective to maintain stability andactivity of an antibody in said antigen-antibody reaction and present ata concentration from about 0.01 to about 20% (w/v); vi) a phenoliccompound inhibitor; v) a protease inhibitor; viii) a protein in anamount effective to immunologically block non-specific antibody bindingand present at a concentration of about 0.05 to 5% (w/v); (b) providinga cylindrical solid phase format contained in a cylindrical vessel, thevessel having an inner wall and at least one open end, said solid phaseformat being circumfrentially and tightly appressed against the innerwall of the vessel, having a pore size of about 20-30 micrometers with aplurality of interstitial spaces and further having immobilized thereinan antigen capable of immunologically recognizing said test substance;(c) contacting the extract of step (a) with solid phase format of step(b); (d) washing the solid phase format of step (c) with a buffer; and(e) detecting said test substance in the test sample by detecting thepresence of said captured primary antibody-test substance complex. 43.The immunoassay of claim 42 wherein said test substance is an antibody.44. The immunoassay of claim 42 wherein said solid phase format isselected from cellulose acetate, polyester coated with polystyrene,cellulose, or nylon.
 45. The immunoassay of claim 44 further comprisinga plurality of said solid phase formats wherein said solid phase formatsare arranged in stacked and contiguous layers wherein each layercaptures a different primary antibody-test substance complex.
 46. Theimmunoassay of claim 44 further comprising a plurality of said solidphase formats wherein said solid phase formats are arranged in stackedand contiguous layers wherein each layer captures a different quantityof said primary antibody-test substance complex.
 47. The immunoassay ofclaim 42 wherein said means of detection is an enzyme.
 48. Theimmunoassay of claim 47 wherein said enzyme is selected from alkalinephosphatase, peroxidase, or β-galactosidase.
 49. The immunoassay ofclaim 47 wherein said enzyme is reacted with a substrate therefore toproduce a detectable insoluble reaction product.
 50. The immunoassay ofclaim 47 wherein said enzyme is reacted with a substrate therefore toproduce a detectable soluble reaction product.
 51. The immunoassay ofclaim 42 wherein said means of detection is a fluorochrome.
 52. Theimmunoassay of claim 51 wherein said fluorochrome is selected fromfluorescein isothiocyanate, tetramethylrhodamine isothiocyanate, Texasred, or phycoerythrin.